Biomedical subjects
Publications and source records attributed to T A Cebula.
The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.
Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains.
Extragenic suppressors of the hisG46 missense mutation were mapped to the 71 and 88 min regions of the Salmonella typhimurium chromosome, positions that in Escherichia coli contain the thrV (tRNA(Thr1)) and thrT (tRNA(Thr3)) genes, respectively. The suppressor loci were identified as mutant alleles of thrV and thrT, using allele-specific colony hybridization. An oligomer, based on the conserved 5' sequence of the thrT and thrV genes in E. coli and designed to contain the putative mutant anticodon, discriminated between suppressor-containing and wild-type strains. Similarly, probes specific for the thrV[SuGGG] and thrT[SuGGG] were used to differentiate the two suppressors. To date, all extragenic suppressors of hisG46 have been identified as either thrV[SuGGG] or thrT[SuGGG]. A near equal distribution of thrV[SuGGG] and thrT[SuGGG] suppressors was found among 29 spontaneous and 43 mutagen-induced hisG46 extragenic suppressor revertants. It was concluded, therefore, that mutant alleles of thrV and thrT are predominantly, if not solely, responsible for intergenic suppression of the hisG46 mutation.
Nitrous acid (NA) is often presumed to cause base substitutions in organisms with double-stranded DNA as a direct consequence of oxidative deamination of adenine and of cytosine residues. Here we summarize evidence indicating that other mechanisms are involved in the case of NA-induced G/C-->A/T transition mutations. We present several models for pathways of NA mutagenesis that may account for our experimental results and overlapping data noted in the literature. One model proposes that the base substitution mutations observed are due to DNA alkylation damage mediated via nitrosation of polyamines and/or other ubiquitous cellular molecules. Other models assume that predisposing lesions, such as G-to-G cross-links, are first formed. The cross-links are pictured as leading to perturbations in DNA structure that allow subsequent opportunity for NA-induced deaminations of cytosine residues in their immediate vicinity. The deaminations preferentially result in G/C-->A/T transition mutations at sites highly dependent on adjoining base sequence context (i.e., in NA "mutational hotspots"). A final model proposes that NA-induced G/C-->A/T transition mutations arise mainly from oxidative deamination of guanosine residues and not from deamination of cytosine residues in duplex DNA.
The mutational specificity of N-methylnitrosourea (MNU), nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), sodium azide (NaN3), 4-nitroquinoline oxide (4NQO), benzo[a]pyrene (BP), nitrofurantoin (NF), aflatoxin B1 (AFB1), adriamycin (ADM) and UVA-activated angelicin in Salmonella typhimurium strain TA100 has been examined using allele-specific oligonucleotide hybridization and DNA sequence analyses. These ten mutagens produced five unique classes of reversion spectra, distinct from spontaneous, or the previously characterized 5-azacytidine, ultraviolet light (UV), 8-methoxypsoralen plus UVA (PUVA) and 60Co-induced mutation spectra. For example, 90% of MNU and MNNG-induced mutations in strain TA100 revertants were G:C-->A:T transitions with the majority (82%) occurring in the first position of the CCC codon. In contrast, NaN3 preferentially induced G:C-->A:T transitions at the second codon position (78%). Although MMS, NQO, BP, NF, ADM and AFB1 induced primarily G:C-->T:A transversions (73-86%), these mutagens fall into two classes based on site preference: NF and AFB1 yielded almost exclusively position two transversions (69-78%) whereas ADM, NQO, BP and MMS exhibited a two-fold preference for site 2 over site 1 (on average 52% versus 22%). Angelicin photomutagenesis resulted in the recovery of G:C-->A:T and G:C-->T:A mutations at both codon positions in roughly equal proportions (approximately 20-25% each). Approximately 1% of the mutagen-induced revertants occurred via extragenic tRNA suppressor mutations, while 1% were multiple (usually tandem double) base substitutions. Ultraviolet mutagenesis experiments demonstrated that tandem base substitutions are promoted by pKM101-encoded mucAB gene products. A comparison of the mutagenic specificity derived for several carcinogens in hisG46 with the responses of several eukaryotic gene targets (e.g. HPRT, aprt, supF) revealed a high concordance between these targets. Thus, the Salmonella hisG46 locus provides a rapid, simple system for determining base substitution specificity and for studying mechanisms of mutagenesis.
PCR was used to introduce a 63-bp deletion into the putative RNA replicase coding sequence of hepatitis A virus. RNA was synthesized in vitro from the deletion mutant cloned into a transcription vector. Upon amplification by PCR, cDNA made from the competitor RNA generated an amplified fragment that could be easily distinguished from the product generated from wild-type hepatitis A virus genomic RNA by gel electrophoresis, when the same primers were used, without further manipulation. The competitor RNA was used as a positive control in PCR-based detection of very low copy numbers of hepatitis A virus genomic RNA in the presence of unrelated hard-shell clam RNA. When the competitor RNA was used for competitive PCR to quantitate wild-type RNA, the presence of one template at a 10-fold to 100-fold higher level almost completely inhibited product formation from the underrepresented template. The competitor RNA should be useful as a control for reverse transcription and PCRs to determine hepatitis A virus genome RNA when accidental contamination of test samples by a wild-type positive control template would compromise the results.
The polymerase chain reaction was used to selectively amplify sequences within the cholera toxin operon from Vibrio cholerae O1. Oysters, crabmeat, shrimp, and lettuce were seeded with V. cholerae and then homogenized or washed with alkaline peptone water, followed by short-term (6- to 8-h) enrichment. A detection limit of as few as 1 V. cholerae CFU per 10 g of food was obtained with amplification reactions from crude bacterial lysates. The method is extremely rapid and obviates the need for DNA isolation from a variety of complex food matrices.
Hepatitis A virus (HAV) is a major cause of infectious hepatitis in humans. In this respect, bivalve mollusks pose a major health concern because they are filter feeders and can concentrate the virus up to 900-fold from contaminated water. Detection of HAV has been hampered because wild-type HAV grows poorly if at all in cell culture. Here we describe a technique for the detection of HAV in shellfish based on reverse transcription coupled with the polymerase chain reaction. RNA is isolated from hard-shell clam tissue and reverse transcribed with avian myeloblastosis virus reverse transcriptase. A portion of the cDNA pool is then amplified with primers specific for HAV. In experiments with an in vitro-synthesized HAV transcript, we were able to detect HAV sequence in the presence of a 200-million-fold excess of shellfish RNA. When intact virus was added to shellfish tissue before the isolation of RNA, the method was capable of detecting 10 viral RNA molecules in a reaction mixture.
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We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.
We investigated the role of the umuDC and samAB operons in the UV mutability of Salmonella typhimurium. umuDC is located on the chromosome, whereas samAB resides on the virulence plasmid pSLT. Using allele replacement and plasmid curing techniques, we found that UV mutability was eliminated when any of three different umuDC alleles (umuD1, umuC1, or umuD1 umuC1) were on the chromosome even when samAB was present. We conclude that samAB normally does not complement umuDC function in S. typhimurium.
Nitric oxide (NO), a multifaceted bioregulatory agent and an environmental pollutant, can also cause genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and intact DNA at physiological pH. That similar DNA damage can also occur in vivo was tested by treating Salmonella typhimurium strain TA1535 with three NO-releasing compounds, including nitroglycerin. All proved mutagenic. Observed DNA sequence changes were greater than 99% C----T transitions in the hisG46 (CCC) target codon, consistent with a cytosine-deamination mechanism. Because exposure to endogenously and exogenously produced NO is extensive, this mechanism may contribute to the incidence of deamination-related genetic disease and cancer.
A rapid method for determining the DNA sequences of Salmonella typhimurium hisD3052 revertants is presented. DNA colony hybridization was used to analyze revertants previously studied by Isono and Yourno [Proc Natl Acad Sci USA 71:1612-1617, 1974]. Synthetic oligodeoxyribonucleotide probes (18-mers) were able to distinguish sequences that differed by a single base pair. Mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (PCR) and directly sequenced. The combined use of DNA-colony hybridization and direct sequencing offers a precise and rapid means for the molecular characterization of hisD3052 revertants.
SOS mutagenesis in prokaryotes is dependent upon the inducible activity of the chromosomally encoded UmuDC proteins, or homologous proteins such as MucAB or ImpCAB which are found on naturally occurring plasmids. Relative to Escherichia coli, however, Salmonella typhimurium is much less responsive to the mutagenic effects of DNA-damaging agents, despite the fact that it possesses both chromosomally and plasmid encoded umu-like operons. In E. coli, activation of the UmuD mutagenesis protein to UmuD' via RecA-mediated proteolysis is a critical step in the mutation fixation pathway. We have used a polyclonal antiserum raised against the E. coli UmuD and UmuD' proteins to show that S. typhimurium expresses cross-reacting material only after treatment with the DNA-damaging agent mitomycin C. The S. typhimurium umuDC operon, therefore, appears to be regulated by mechanisms similar to the E. coli umuDC operon. After induction, the S. typhimurium UmuD protein was processed to UmuD' in both S. typhimurium and E. coli. However, the S. typhimurium UmuD protein appears to be cleaved more efficiently than the E. coli UmuD protein under similar conditions. The data suggest that conversion of UmuD to the mutagenically active UmuD' is not the rate-limiting factor accounting for the weakly mutable phenotype of S. typhimurium.
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An improved DNA colony-hybridization method for the rapid characterization of Salmonella typhimurium hisG46 revertants is described. Oligodeoxyribonucleotides (15-mers) complementary to each of 6 possible transition or transversion mutations and an extragenic suppressor mutation, underlying the His+ phenotype, were prepared. Optimal sequence discrimination was achieved by hybridizing 15-mers at the apparent dissociation temperature (Td) for 2 h with chromosomal DNA of revertant colonies affixed to Whatman 541 filters. Subsequent exposure of filters to UVA radiation (320-400 nm) in the presence of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) resulted in cross-linking of perfectly matched probes and target DNA sequences while sequences containing a single base-pair mismatch could be discriminated with a brief denaturing wash. No false negative results were obtained with the new procedure. An analysis of 204 spontaneous and 174 PUVA-induced TA100 revertants is presented.
In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.
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